Gammaherpesviruses result in lifelong infection associated with malignancies and other chronic disease in immune deficient individuals. The human gammaherpesviruses include Epstein Barr virus and Kaposi's Sarcoma associated virus, which are associated with Burkitt's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorder, nasopharyngeal carcinoma, peritoneal effusion lymphoma and Kaposi's sarcoma. Given the strict host specificity of the human gammaherpesviruses, a major challenge is to understand the host and viral factors that regulate the outcome of gammaherpesvirus infection in vivo, in both healthy and immune compromised individuals. This proposal makes extensive use of the mouse gammaherpesvirus 68, to investigate the genetic contribution of viral and host genes in shaping the outcome of infection. Through our previous work, we developed an extensive knowledge of the precise in vivo contexts in which a viral gene, the viral cyclin, promotes chronic infection. We identified seven different parameters of gammaherpesvirus infection and disease, including models of immune deficiency that are dependent on the virus encoded cyclin homolog. In this grant, we will dissect the molecular pathways used by the viral cyclin to promote infection in vivo, using recombinant viruses in which the viral cyclin has been replaced by alternate viral or cellular cyclins. We will characterize the viral cyclin activity in all cyclin dependent aspects of infection, identify the infected cell reservoir in presence and absence of the viral cyclin, and define the molecular mechanism of the viral cyclin in both persistent infection and reactivation from latency infection. These recombinant viruses reveal distinct, non-overlapping mechanisms of viral cyclin action. Moreover, since different cellular cyclins are able to substitute for distinct phases of the viral infection, comparing the biochemical properties of different cyclins in distinct contexts of infection will result in significant insights in gammaherpesvirus infection and disease. By integrating our unique in vivo insights into the viral cyclin with these recombinant viruses and our identification of the cellular target of the viral cyclin in virus reactivation, this grant wil combine biochemistry and genetics, with in vitro and in vivo models of infection to define new molecular pathways that promote gammaherpesvirus infection and pathogenesis in the whole animal. Further, we propose to test candidate inhibitors of viral cyclins and viral cyclin dependent functions both in vitro and in vivo. Finally, our demonstration that host cyclins can facilitate virus infection and that a specific host tumor suppressor can repress virus reactivation promises that our investigation of the virus encoded cyclins will advance the field of cancer biology beyond virus associated oncogenesis. These studies of the v-cyclin have, and will continue to yield fundamental insights into gammaherpesvirus infection and further elucidate new mechanisms by which gammaherpesvirus pathogenesis can be abrogated. PUBLIC HEALTH RELEVANCE: The cellular cyclins have been strongly associated with tumorigenesis since their discovery, and while they were initially considered exclusively for regulating cell proliferation, recent studies show that frequent overlap in the cell proliferation functions, yet that their roles in cell fate, development and differentiation are distinct. Our studies of the virus encoded cyclins have identified that these cyclins are required at several stages of infection and pathogenesis, and that they work by at least two different mechanisms and that one of these mechanisms functions to oppose the activity of a cellular tumor suppressor protein. The work proposed here will define the mechanism of the viral cyclins during authentic infection and disease and will identify potential interventions relevant to human inflammatory disease and malignancies, both those associated with gammaherpesvirus infection and related to cellular cyclins and tumor suppressors.